Peripheral nerve injuries are common and associated with devastating morbidity. Direct nerve repair without a graft is rarely possible and many techniques have been used to bridge the gap. However, nerve injuries requiring long nerve grafts or constructs result in poor functional outcomes. All graft constructs (i.e. autografts, cadaveric allografts, acellular nerve allografts, and nerve conduits) have length limits for supporting regeneration. As the length of the graft construct increases, regeneration decreases until few or no axons reach the distal nerve. Our research demonstrates long nerve grafts accumulate significant quantities of senescent Schwann cells (SenSCs), with the presence of SenSCs preceding, not following, diminished axonal regeneration. Our recent data demonstrates SenSCs cause reduced axonal regenerative capacity in cell culture and in vivo. This data also suggests SenSCs may exert their effects on axons through the expression of Notch ligands. We hypothesize SenSCs impact axonal regeneration across nerve grafts. To test this hypothesis, we utilize animal models of nerve grafts that allow us to modify graft components including SenSC quantity, vascular reperfusion time, and Notch-ligand expression in both SenSCs and normal SCs. We determine how modifying these components impacts axonal regeneration across nerve grafts. The goal of this proposal is to establish the impact of SenSCs on axonal regeneration and functional recovery in long nerve grafts and provide a mechanism for reduced axonal growth due to SenSCs. We first determine the impact of SenSC quantity on axonal regeneration across nerve grafts. We then determine how improving vascular reperfusion (decreased vascular reperfusion time) affects axonal regeneration across nerve grafts and the accumulation of SenSCs. Our preliminary data identified increased expression of Notch ligands in long nerve grafts compared to short nerve grafts concomitant with decreased axonal regeneration and increased SenSC accumulation. As such, we determine the impact of Notch ligand expression by SenSCs on axonal regeneration across nerve grafts using overexpression and RNAi techniques targeting Notch ligand expression. The proposed plan will improve our understanding as to why axonal regeneration is reduced or fails in long nerve grafts and may lead to therapies, including novel therapeutic targets. The specific aims of this proposal are: Aim 1: Determine the impact of SenSCs in limiting regeneration across nerve grafts; Aim 2: Determine the impact of SenSC-mediated Notch ligand expression on axonal growth.